COUPLING OF AN ENDOGENOUS 5-HT1B-LIKE RECEPTOR TO INCREASES IN INTRACELLULAR CALCIUM THROUGH A PERTUSSIS-TOXIN-SENSITIVE MECHANISM IN CHO-K1 CELLS

1 Chinese hamster ovary cells (CHO-K1) express an endogenous 5-hydroxytryptamine (5-HT)(1B)-like receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin (PTX)-sensitive mechanism. Furthermore, the human adenosine A(1) receptor when expressed in CHO-K1 cells (CHO-A1) has been shown to mobilize intracellular Ca2+ through a PTX-sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5-HT1B-like receptor was able to stimulate increases in intracellular free [Ca2+] ([Ca2+](i)) in CHO-A1 cells. 2 In agreement with previous studies using CHO cells, 5-hydroxytryptamine (5-HT) elicited a concentration-dependent inhibition of forskolin-stimulated [H-3]-cyclic AMP production in CHO-A1 cells (p[EC(50)] = 7.73 +/- 0.13). 5-HT (1 mu M) inhibited 47 +/- 5% of the [H-3]-cyclic AMP accumulation induced by 3 mu M forskolin. Forskolin stimulated [H-3]-cyclic AMP accumulation was also inhibited by the 5-HT1 receptor agonists (p[EC(50)] values) 5-carboxyamidotryptamine (5-CT; 8.07 +/- 0.08), RU 24969 (8.12 +/- 0.33) and sumatriptan (5.80 +/- 0.31). 3 5-HT elicited a concentration-dependent increase in [Ca2+](i) in CHO-A1 cells (p[EC(50)] = 8.07 +/- 0.05). In the presence of 2 mM extracellular Ca2+, 5-HT (1 mu M) increased [Ca2+](i) from 174 +/- 17 nM to 376 +/- 22 nM. The 5-HT1 receptor agonists (p[EC(50)] values), 5-carboxyamidotryptamine (5-CT; 7.9 +/- 0.02), RU 24969 (8.1 +/- 0.07) and sumatriptan (5.9 +/- 0.11) all elicited concentration-dependent increases in [Ca2+](i). Similar maximal increases in [Ca2+](i) were obtained with each agonist. The selective 5-HT1A receptor agonist, 8-OH-DPAT (10 mu M) did not stimulate increases in [Ca2+](i). 5-HT (100 mu M) and 5-CT (10 mu M) did not stimulate a measurable increase in [H-3]-inositol phosphate accumulation in CHO-A1 cells. 4 5-HT (1 mu M)-mediated increases in [Ca2+](i) were insensitive to the 5-HT receptor antagonist, ritanserin (5-HT2; 100 nM), ketanserin (5-HT2; 100 nM), LY-278,584 (5-HT3; 1 mu M) and WAY 100635 (5-HT1A; 1 mu M). The response to 5-HT (100 nM) was antagonized by the non-selective 5-HT1 antagonist, methiothepin (pK(b) = 8.90 +/- 0.09) and the 5-HT1D antagonist GR 127935 (pK(b) = 10.44 +/- 0.06). 5 Pretreatment with PTX(200 ng ml(-1) for 4 h) completely attenuated the Ca2+ response to 100 mu M 5-HT. 6 In untransfected CHO-K1 cells, 5-HT (1 mu M), RU 24969 (1 mu M), and 5-CT (1 mu M) elicited increases in [Ca2+](i) similar to those observed in CHO-A1 cells. 7 These data demonstrate that in CHO-K1 cells the endogenously expressed 5-HT1B-like receptor couples to the phospholipase C/Ca2+ signalling pathway through a PTX-sensitive pathway, suggesting the involvement of G(i)/G(o) protein(s).